TET2 (ten-eleven translocation (TET) oncogene family member 2) is emerging as an important tumor suppressor in a variety of cancers, including in leukemia. Through three consecutive oxidation reactions, TET2 converts 5-methylcytosine into 5-hydroxymethycytosine (5hmc), 5-formylcytosine, and 5-carboxylcytosine. 5- carboxylcytosine can then be converted to cytosine by the base excision repair pathway or during normal DNA replication, resulting in base demethylation. The current paradigm is that loss of function TET2 mutations results in DNA hyper-methylation, leading to decreased tumor suppressor gene expression and tumor development. However, we currently have a poor understanding of the nature of genes and regulatory elements that are under the control of TET2 and the ways in which TET2 may be targeted to DNA. Preliminary data presenting here comparing the epigenome of leukemic cells harboring wild-type versus TET2 mutations indicate that loss of TET2 function does not alter global methylation, but methylation of specific non-CpG island enhancer elements. As TET2 does not harbor a sequence specific DNA binding domain, it is hypothesized that recruitment to these enhancer elements is dependent on associations with specificity factors that interact with DNA in a sequence specific manner. The goal of experiments outlined in this R03 proposal is identify such proteins. We will then determine how depletion of these factors alters TET2-DNA interactions, genome wide 5hMC modification, and gene expression. Results from these experiments will advance our understanding of DNA methylation in general and specifically of TET2, one of the most frequently mutated genes in hematologic malignancies.